WO2007071449A1 - Soluble gp130 molecule variants useful as a medicament - Google Patents
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Definitions
- Soluble gpl30 molecule variants useful as a medicament are useful as a medicament.
- a polypeptide-dimer built of two identical monomeric fragments comprising the domains 1 to 3 of the extracellular (soluble) part of glycoprotein (gp) 130 and a particular polypeptide spacer are described, which are covalently linked with each other and which bear significant advantages concerning their production rate in host cells, their improved purification and their potential to bind to IL-6/soluble IL-6 receptor complexes. Furthermore, a pharmaceutical composition containing said dimeric molecule and various medical uses are described.
- IL-6 pleiotropic cytokine interleukin-6
- IL-6R soluble IL-6 receptor
- gp80 soluble IL-6 receptor
- IL-6R Since the cytoplasmic domain of IL-6R lacks kinase activity, signalling by gpl30 homodimer can be induced by IL-6 in complex with membrane-bound or soluble IL- 6R. While gpl30 can be found on nearly every cell type, IL- 6R expression is restricted to hepatocytes and leukocytes. However, these cells can be activated by IL-6 via sIL-6R which is released from IL- 6R expressing cells either by proteolytic cleavage or alternative splicing. This mechanism is called trans- signalling. Indeed, several cellular activities have been shown which are dependent on the complex of sIL-6R plus IL-6 and are not inducible by IL-6 alone.
- Trans-signalling plays a key role in the switch from innate to acquired immune responses and is crucial for the clearance of neutrophilic infiltrates and the recruitment and activation of T and B cells for a sustained immunological answer. Dysregulation of this mechanism leads to the development and persistence of chronic and/or allergic inflammatory disease such as Crohn's disease, rheumatoid arthritis, asthma and others .
- the efficiency of the new molecule sgpl30(Dl- 3) Fc to bind to IL-6/sIL-6R was optimized by fusing polypeptide spacers of different length between the sgpl30(Dl- 3) and the IgG-Fc part.
- the binding skills of the resulting molecules were determined by specific enzyme-linked immunosorbent assays (ELISA) and compared to the parental molecule sgpl30Fc ( Figure 5) .
- Binding of IL-6/sIL-6R complexes has been shown to inhibit the anti-apoptotic effect of IL-6 on monocytes in Crohn's disease patients indicating that such compound is useful for the treatment of said disease and related diseases such as colitis, rheumatoid arthritis, psoriasis, peritonitis and others .
- Crohn's disease is a chronic inflammatory disorder of the entire gastrointestinal tract characterized by frequently occurring relapses of acute inflammation. Inflammation associated with infection, injury, and other factors rapidly induces the acute phase reaction (APR) accompanied by the expression of acute phase proteins (APPs) .
- APR acute phase reaction
- APPs acute phase proteins
- the APR mainly results in an increase of vascular permeability and fever.
- IL- 6 family members upregulate the expression of type II APP genes which is mediated by the (STAT3) . Strong activation of STAT3 (i.e. tyrosine phosphorylation) has been described in colonic tissues from inflammatory bowel disease (IBD) patients.
- sgpl30 (Dl-3 ) Fc was expressed at higher amounts in eukaryotic cells than sgpl30Fc, generated less undesired side products and was separable from remaining impurities by standard column chromatography.
- Figure 1 Schematic drawing of sgpl30 (D1-D3 ) S n molecules sgpl30 (D1-D3 ) S n variants are generated by deletion of the domains D4 to D6 of the parental molecule sgpl30Fc and their replacement by a polypeptide spacer of various length (S n ) . "n" indicates the number of repeats within the spacer region. Subsequently the molecule is covalently dimerized either by an IgG-Fc fragment (upper panel) to build sgpl30 (D1-D3) S n -Fc or
- these molecules can contain sequence tags for purification and/or detection purposes .
- FIG. 2 Expression of sgpl30 (D1-D3) S 11 in CHO cells
- the expression of sgpl30 (D1-D3 ) S n is exemplarily shown for sgpl30 (D1-D3) Si-Fc.
- a respective construct pDEST40_sgpl30 (Dl- DS)S 1 -Fc
- pDEST40_sgpl30 (Dl- DS)S 1 -Fc was transiently transfected into CHO cells.
- another set of CHO cells was transfected with a construct comprising the parental sgpl30Fc.
- the cells were incubated for 24 h, the supernatants were collected, and the sgpl30 fusion proteins were immunoprecipitated.
- the precipitates were finally analyzed by SDS-PAGE and detected by using an anti -human IgG antibody according to standard Western blot procedures .
- FIG. 3 Purification of sgpl30Fc fusion molecules Size exclusion chromatography (SEC) of sgpl30Fc.
- the upper panel shows the elution profile (chromatogram) derived during gel filtration by UV scanning.
- the middle and lower panels represent the analysis of the collected fractions by native polyacrylamide gel electrophoresis (PAGE) and staining with either coomassie (COOM) or silver according to standard procedures .
- SEC Size exclusion chromatography
- Figure 5 Specific binding of IL-6/sIL-6R complexes by sgpl30 (D1-D3) S n -Fc molecules
- Figure 6 Schematic drawing of an assay to detect sgpl30 (Dl- D3) S n -Fc binding antibodies
- sgpl30 (D1-D3) S n -Fc fixed to a solid matrix is incubated with an antibody mixture (e.g. contained in serum samples) which was previously labeled (e.g. by fluorescence dyes). Unbound antibodies are washed away and specific binding molecules are detected by state-of-the-art technologies.
- sgpl30 (D1-D3 ) S n -Fc fixed to a solid matrix is incubated with an antibody mixture (e.g. contained in serum samples) .
- Unbound antibodies are washed away and the remaining sgpl30 (Dl -D3 ) S n -Fc /antibody complex are subsequently incubated with a certain amount of labeled sgpl30 (D1-D3) S n -Fc (e.g. fluorescence dyes) and are finally detected by state-of-the-art technologies.
- sgpl30 Dl -D3
- S n -Fc e.g. fluorescence dyes
- Figure 7 cDNA sequence of optimized sgpl30 (D1-D3) The sequence includes the signaling peptide as well as the domains Dl to D3 of sgpl30. The sequence was codon optimized for the expression of the encoded polypeptide in mammalian cells .
- Figure 8 Amino acid sequence of soluble gpl30 (D1-D3)
- the present invention relates to a polypeptide-dimer of two monomeric fragments, wherein the monomeric fragments comprise domains 1 to 3 (Dl to D3) of the extracellular part of glycoprotein (gp) 130 and at their C-terminal ends a polypeptide spacer having a length of 5 to 30 amino acids, wherein both monomeric fragments are covalently linked to each other, wherein the spacer length determines optimal binding of the resulting dimeric protein to the IL-6/soluble IL-6 receptor complex and wherein said polypeptide-dimer exhibits a significantly reduced potential to build homomeric aggregates and molecule fragments, and wherein significantly higher productivity in host cells is obtained.
- the monomeric fragments comprise domains 1 to 3 (Dl to D3) of the extracellular part of glycoprotein (gp) 130 and at their C-terminal ends a polypeptide spacer having a length of 5 to 30 amino acids, wherein both monomeric fragments are covalently linked to each other, wherein the spacer length determines optimal binding of the
- the two monomeric fragments of the polypeptide-dimer of the present invention are identical.
- the dimer does not contain further domains (D4 to D6) of sgpl30.
- the linkage of two monomers to build the dimer can be carried out by a person skilled in the art by well known methods.
- the two soluble gpl30 molecules can be linked to each other via one or more disulfide bridges. This can be achieved, e.g. by recombinant expression, wherein the nucleic acid sequence encoding sgpl30 is fused at its C- terminus to a polypeptide linker which contains one or more codons encoding cystein residues between the C-terminus of sgpl30 and the stop-codon.
- the two soluble gpl30 molecules may be C-terminal fused to an IgG-Fc fragment including hinge region (either directly or via a linker) .
- free cystein residues within the hinge region of the Fc molecule may be deleted by mutation to reduce the risk of building undesired intermolecular disulfide bridges.
- the molecules of the dimer may be tagged, e.g.
- His-His-His-His-His-His-His His 6 ) , Myc , Strep, polyarginine, Flag, green fluorescence protein (GFP), TAP, glutathione S-transferase (GST) , HA, calmodulin-binding peptide (CBP), maltose-binding protein (MBP), V5 , HSV, S, vesicular stomatitis virus (VSV) , Protein C, Luciferase, GIu- GIu, E, beta-GAL, T7 or other epitopes to which antibodies or other binding molecules are available to allow purification by suitable chromatography systems and/or detection, e.g. by westernblot, ELISA, bioassays etc.
- the sgpl30 subunits are fused at their C-terminal end to (poly) peptide spacers having a length of 5 to 30 amino acids, preferably (i) 10 to 25 or (ii) 15 to 25 amino acids or, particularly preferred, 10 to 15 amino acids.
- amino acids building the spacer is not particularly critical, however, amino acids (like G or S) are preferred which (a) ensure ideal steric flexibility allowing an optimum alignment of the monomers, (b) are not easily accessible by proteases and antibodies and (c) are not charged in order to reduce or eliminate any undesired aggregation of the dimer with other molecules or the formation of trimers, etc..
- Preferred amino acids for building the spacer molecule are one or more repeats of the amino acid sequence "GGGGS" but the spacer can comprise any other sequence which enhances ligand binding of said sgpl30 molecule.
- the polypeptide-dimer of the present invention forms less than 50% of homomeric aggregates and molecule fragments, preferably less than 25% of homomeric aggregates and molecule fragments, more preferably less than 10% of homomeric aggregates and molecule fragments and, most preferably, less than 5% of homomeric aggregates and molecule fragments.
- the present invention also provides polynucleotides encoding a polypeptide-dimer (or the respective monomers) of the present invention, which can be codon optimized for the efficient production of the encoded protein in eukaryotic host cells, bacteria, yeast or insect cells.
- said polynucleotide comprises the nucleic acid sequence shown in Figure 7.
- the present invention also relates to expression vectors containing a polynucleotide of the invention and corresponding host cells.
- a variety of means can be used to generate and identify mutations or modifications of sgpl30 that have the desired properties.
- Site-directed mutagenesis by standard techniques of the DNA encoding sgpl30 or state-of-the-art methods such as restriction digest and ligation may be used, followed by analysis of the collection of products to identify mutated molecules having the desired properties.
- the recombinant vectors can be constructed according to methods well known to the person skilled in the art, e.g., Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Habor Laboratory (1989) N. Y.
- a variety of expression vector/host systems may be utilized to contain and express sequences encoding the sgpl30 polypeptides of the present invention. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g.
- virus expression vectors e.g., cauliflower mosaic virus, CaMV, tobacco mosac virus, TMV
- bacterial expression vectors e.g. Ii or pBR322
- control elements are those non-translated regions of the vector-enhancers, promoters, 5'- and 3 ⁇ -untranslated regions which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the Bluescript . RTM. phagemid (Stratagene, LaJoIIa, CA) or pSPORTl.TM plasmid (Gibco BRL) and the like may be used.
- the baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO; and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from- mammaliangenes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding the sgpl30 polypeptides, vectors based on SV40 or EBV may be used with an appropriate selectable marker.
- Promoters or enhancers derived from the genomes of plant cells e.g., heat shock, RUBISCO; and storage protein genes
- plant viruses e.g., viral promoters or leader sequences
- a number of expression vectors may be selected depending upon the use intended for the polypeptide dimer of the present invention.
- Vectors suitable for use in the present invention include, but are not limited to the pSKK expression vector for expression in bacteria.
- yeast Saccharomyces cerevisiae
- a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used; for reviews, see Grant et al . (1987) Methods Enzymol . 153 :516-544.
- sequences encoding the antibody of the present invention may be driven by any of a number of promoters.
- viral promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV (Takamatsu, N (1987) EMBO J. £: 307-311) .
- constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (see, for example Hobbs, S. and Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, NY; pp. 191-196.
- An insect system may also be used to express the sgpl30 molecules of the present invention.
- Autographs California nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
- the sequences may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter.
- Successful insertion of the gene encoding sgpl30 will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
- the recombinant viruses may then be used to infect, for example S.
- frugiperda cells or Trichoplusia larvae in which APOP may be expressed (Engelhard, E. K. et al . (1994) Proc . Nat. Acad. Sci . SKL:3224-3227) .
- a number of viral-based expression systems may be utilized.
- sequences encoding the polypeptide (s) of the present invention may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non- essential El or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing the protein in infected cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 8JL: 3655-3659).
- transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells .
- RSV Rous sarcoma virus
- HACs Human artificial chromosomes
- HACs may also be employed to deliver larger fragments of DNA than can be contained and expressed in a plasmid.
- HACs of 6 to 1OM are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes .
- Specific initiation signals may also be used to achieve more efficient translation. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding the sgpl30, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in case where only coding sequence is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic.
- Enhancers which are appropriate for the particular cell system which is used, such as those described in the literature (Scharf, D. et al . (1994) Results Probl . Cell Differ. 20:125-162).
- a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed polypeptide chains in the desired fashion.
- Post-translational processing which cleaves a "prepro" form of the polypeptide may also be used to facilitate correct insertion, folding and/or function.
- Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, or W138), are available from the American Type Culture Collection (ATCC; Bethesda, MD) and may be chosen to ensure the correct modification and processing of the foreign polypeptide chains. For long-term, high-yield production of recombinant polypeptides, stable expression is preferred.
- cell lines which stably express sgpl30 chains may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched medium before they are switched to selective media.
- the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stable transformed cells may be proliferated using techniques appropriate to the cell type.
- the host cells After the introduction of the recombinant vector (s), the host cells are grown in a selective medium, which selects for growth of vector-containing cells. Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler, M. et al . (1977) Cell 11:223-232) and adenine phosphoribosyltransferase (Lowy, I. et al . (1980) Cell 2 ⁇ :817-823) genes which can be employed in tk.sup. or aprt . sup . -cells, respectively.
- the herpes simplex virus thymidine kinase Wigler, M. et al . (1977) Cell 11:223-232
- adenine phosphoribosyltransferase Liowy, I. et al . (1980) Cell 2 ⁇ :817-8
- antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler, M. et al . (1980) Proc . Natl. Acd. Sci . 7_7: 3567-3570) ; npt , which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al . (1981) J. MoI. Biol. 150:1-14) and als or pat, which confer restistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra) .
- trpB which allows cells to utilize indole in place of tryptophan
- hisD which allows cells to utilize histinol in place of histidine
- Purification of the recombinant polypeptides is carried out by any one of the methods known for this purpose, i.e., any conventional procedure involving extraction, precipitation, chromatography, electrophoresis, or the like.
- a further purification procedure that may be used is affinity chromatography using monoclonal antibodies or other molecules which bind the target polypeptide and which are produced and immobilized on a gel matrix contained within a column.
- Impure preparations containing the recombinant polypeptide are passed through the column or the matrix will be used in a batch technology to bind target polypeptides. The polypeptide will be bound to the matrix while the impurities will not. After washing the polypeptide is eluted from the matrix by a change in pH or ionic strength.
- polypeptide-dimers of the present invention are useful in the treatment and/or prevention of all pathologies, in which the activity of the agonistic IL-6/sIL-6R complex contributes to the pathogenesis of a disease and must be inhibited.
- therapeutic uses of the polypeptide-dimers of the present invention would include the following:
- IL- 6 appears to be directly involved in multiple myeloma by acting in either an autocrine or paracrine fashion to promote tumor formation. Furthermore, the elevated IL- 6 levels create undesirable secondary effects such as bone resorption, hypercalcemia and cachexia. In these cases it is known that sIL-6R sensitizes target cells for IL-6. Therefore, the polypeptide-dimers of the invention as described herein would be beneficial for both the secondary effects as well as inhibiting tumor growth.
- autoimmune diseases the pathogenic significance of IL-6 in autoimmune disorders has been reviewed by many authors in the literature (see, e.g. Yoshizaki et al . (1992) Semin. Immunol.
- interference with IL-6 signal transduction may be useful for autoimmune disease therapy (Nishimoto et al . (1999) Intern. Med. 38 (2) :178-182) .
- autoimmune disease therapy Nakamoto et al . (1999) Intern. Med. 38 (2) :178-182
- pathologies are systemic lupus erythematosus, Hashimoto's thyroiditis, scleroderma, rheumatoid arthritis, multiple sclerosis, autoimmune epithelitis, diabetes mellitus, Sjogren's syndrome, polymyositis, glomerulonephritis and other inflammatory diseases, such as psoriasis, Crohn's disease, ulcerative colitis and uveitis.
- inflammation- associated cancer diseases such as colon cancer are mentioned.
- IL-6 appears to be critical mediator of osteoclastogenesis, leading to bone resorption. Importantly, IL-6 only appears to play a major role in the estrogen-depleted state, and apparently is minimally involved in normal bone maintenance. Consistent with this, experimental evidence indicates that function-blocking antibodies to IL-6 can reduce the number of osteoclasts. While estrogen replacement therapy is also used, there appear to be side effects that may include an increased risk of endometrial and breast cancer. Thus, the polypeptide-dimers of the present invention would be more specific to reduce osteoclastogenesis to normal levels.
- IL-6 may be a mediator of tumor necrosis factor (TNF) that leads to cachexia associated with AIDS and cancer, perhaps by reducing lipoprotein lipase activity in adipose tissue. Accordingly, the polypeptide-dimers of the invention described herein would be useful in alleviating or reducing cachexia in such patients.
- Bacterial and viral infections the presence of Human Herpes Virus (HHV8) has been demonstrated in more than 91% of Karposi ' s sarcoma (KS) lesions. Moreover, the virus has been identified in primary effusion lymphoma (PEL) and in patients with multicentric Castleman's disease (MCD) .
- PEL primary effusion lymphoma
- MCD multicentric Castleman's disease
- MM multiple myeloma
- HHV8 bone marrow dendritic cells from multiple myeloma (MM) patients were shown to be infected by HHV8. Since then, the association of HHV8 with MM has been a subject of fierce debate, which was recently revived.
- the genome of HHV8 codes for several proteins with significant homologies to human anti-apoptotic proteins, chemokines, and cytokines including a vital form of viral IL-6 (vIL-6) with 25% homology to human IL-6.
- vIL-6 has been demonstrated to have biologic activities reminiscent of human IL-6, i.e. stimulation of proliferation of murine hybridoma and human myeloma cells.
- vIL-6 played an important role in the pathogenesis of HHV8 -associated disorders.
- the contribution of the IL-6R to vIL-6 signaling has been discussed controversially.
- One group using unpurified supernatants of vIL-6 transfected COS-7 cells has shown that STAT activity was induced in cells expressing gpl30 but no IL- 6R.
- another group found that the activity of vIL-6 was reduced by an IL- 6R antagonist, arguing for an involvement of IL-6R in vIL-6 signaling.
- the present invention also relates to a pharmaceutical composition containing an effective amount of a polypeptide- dimer or polynucleotide of the present invention, preferably combined with a pharmaceutically acceptable carrier.
- a pharmaceutically acceptable carrier is meant to encompass any carrier, which does not interfere with the effectiveness of the biological activity of the active ingredient and that is not toxic to the host to which it is administered.
- suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.
- Such carriers can be formulated by conventional methods and can be administered to the subject at an effective dose.
- an “effective amount” refers to an amount of the active ingredient that is sufficient to affect the course and the severity of the disease, leading to the reduction or remission of such pathology.
- an "effective dose” useful for treating and/or preventing these diseases or disorders may be determined using methods known to one skilled in the art (see for example, Finl et al . (1975) The Pharmacological Basis of Therapeutics, Goodman and Gilman, eds . Macmillan Publishing Co., New York, pp. 1-46) .
- compositions may be effected by different way, e.g. by intravenous, intraperetoneal , subcutaneous, intramuscular, topical (e.g. enema, inhalation, salve, drops) or intradermal administration.
- the route of administration depends on the kind of therapy and the kind of compound contained in the pharmaceutical composition.
- the dosage regimen will be determined by the attending physician and other clinical factors. As is known in the medical arts, dosages for any one patient depends on many factors, including the patients size, body surface area, weight, age, sex, the particular compound to be administered, time and route of administration, the kind of therapy, general health and other drugs being administered concurrently.
- Preferred medical uses of the polypeptide-dimers and polynucleotides of the present invention described above are the treatment of bone resorption, hypercalcemia, cachexia, tumors, cancer, autoimmune diseases, inflammatory diseases such as Crohn's disease, ulcerative colitis, rheumatoid arthritis, juvenile rheumatoid arthritis, asthma, psoriasis, lupus erythematosus, multiple sclerosis, uveitis and others, bacterial or viral infections.
- the Gateway cloning system components (AccuPrime Pfx DNA Polymerase, the donor vector pD0NR221, the CMV promoter-controlled expression vector pcDNA-
- DEST40, BP and LR recombinase for insert transfer and competent E. coli cells were purchased from Invitrogen
- the QuikChange II site-directed mutagenesis kit was obtained from Stratagene (Amsterdam, The Netherlands) .
- HYPUR purified mutagenesis primers were from MWG
- CHO-Kl cells were obtained from the German Collection of Microorganisms and Cell Cultures
- a mouse anti -human IgG (Fc) monoclonal antibody was used (CBL102; Chemicon; Hofheim, Germany) .
- Western blot secondary detection was performed with a anti -mouse IgG HRP-linked antibody, ECL-Plus Western blotting substrate and Hyperfilm ECL (all from GE Healthcare; Freiburg, Germany) .
- Roller bottles (2.1 L, 2 , 5X surface) were purchased from Greiner Bio-One (Frickenhausen, Germany) .
- the domains D4-D6 of sgpl30Fc were replaced by ideal spacer elements (Si, S 2 , S 3 and S 5 ) using a primer pair which bridged the end of domain D3 (YED) and the beginning of the Fc part (SCD) of sgpl30Fc and encoded a different number of repeats of the amino acid spacer sequence "glycin-glycin-glycin-glycin-serin" (GGGGS).
- the variant sgpl30 (D1-D3) Fc was left without spacer (for clarification purposes: if a variant "sgpl30 (D1-D3) S 0 -Fc" is mentioned this means that the molecule does not contain a spacer peptide and therefore is identical to sgpl30 (D1-D3) Fc) .
- Positive clones were identified by restriction digest with AlwNI and verified by complete sequencing as described above. Subsequently, the insert was transferred to the expression vector pcDNA-DEST40 by Gateway LR recombination.
- CHO-Kl cells were grown in Ham' s F12 medium supplemented with 10% FBS at 37°C and 5% CO 2 in a water-saturated atmosphere. Maintenance cultures were split every 3-4 days and used only up to 20 passages. Cells were transfected with the expression construct pcDNA-DEST40_sgpl30 (D1-D3) S n -Fc using Lipofectamine 2000 and standard conditions for CHO-Kl supplied by Invitrogen. For a first transient expression test, CHO-Kl were transfected in 6-well plates, and both cells and supernatants were harvested 24 h after transfection.
- sgpl30 (D1-D3 ) S n -Fc was immunoprecipitated from the supernatants using Protein A/G Plus Agarose and the anti-human IgG (Fc) antibody according to the manufacturer's instructions.
- Whole cell protein was extracted and Western blots with anti -human IgG (Fc) antibody were performed with the cell lysates and immunoprecipitates as described in Waetzig et al . , J. Immunol.168: 5342 (2002).
- CHO-Kl cells were transfected and selected using 400 ⁇ g/ml G418 in 10-cm plates.
- a pre-selected polyclonal CHO-Kl pool was transferred to roller bottles.
- Supernatants of the confluent cells were harvested three times a week, centrifuged twice at 4,000 rpm and 4 0 C for 15 min to remove cell debris and either processed immediately or frozen at -80 0 C.
- stable cell clones were selected from a pre-selected pool using the limited dilution method and characterized by Western blot expression analysis as described above. The clone with the highest and most stable expression was transferred to roller bottles and used for further production.
- sgpl30 (D1-D3) S n -Fc (exemplarily shown for sgpl30 (D1-D3) Si-Fc) was expressed at production rates which were even better than those obtained for the parental sgpl30Fc protein. This indicates that the expression of a smaller variant of sgpl30Fc appears to be enhanced and confers advantages for the preparation of the protein. Finally, higher production rates will significantly lower the costs for the industrial production.
- Cellulose acetate filters (0.45 ⁇ m) for a vacuum filter unit were purchased from Sartorius (G ⁇ ttingen, Germany) .
- PBS was from PAA Laboratories (C ⁇ lbe, Germany) .
- Materials for affinity and size exclusion chromatography (SEC) were all obtained from GE Healthcare (Freiburg, Germany) : MabSelect material (product code 17-5199-01) in a XK16/20 column, PD-10 desalting columns and HiLoad 26/60 Superdex 200 pg for SEC.
- Amicon Ultra-15 50 kD Ultracel-PL membrane concentration units were purchased from Millipore (Eschborn, Germany) . Ready-made acrylamide-bis solution (19:1, 30%) for PAGE was supplied by Bio-Rad (Munich, Germany) .
- sgpl30 (D1-D3) S n -Fc variants The purification of the sgpl30 (D1-D3) S n -Fc variants is exemplarily shown for sgpl30 (D1-D3) S 1 -Fc .
- sgpl30 (D1-D3) Si-Fc- containing supernatants from roller bottle cultures were purified at 4°C using a P-I peristaltic pump and a AKTA Purifier 100 System (both from GE Healthcare; Freiburg, Germany) . The protocol was based on the manufacturer's recommendations for the purification of monoclonal antibodies. After centrifugation, the pH of the fresh or thawed (on ice) supernatant was adjusted to 6.7-7.0.
- the supernatant was degassed and - if necessary - pH was adjusted again to a value of 6.7-7.0.
- the PBS-equilibrated affinity chromatography column (6-25 ml MabSelect in a XK16/20 column) was loaded with 2-4 L of supernatant at a flow rate of 3-10 ml/min using the P-I pump. After washing with PBS, the column was transferred to the AKTA purifier and washed again with PBS until the A 28 o stabilized after quantitative removal of unbound protein.
- the AKTA system was equipped with two 50 mM sodium citrate buffers at pH 3.25 and 5.5, respectively, which were mixed to produce the desired pH conditions.
- One washing step at pH 5.1 was followed by elution with pH 3.7.
- Fractions of 10 ml were collected in 15-ml tubes containing 2 ml 1 M Tris-HCl (pH 11) .
- the peak fractions were pooled, and the pH was measured and adjusted to 7.5, if necessary.
- Pool protein concentration was measured by A 28 o and the pool was carefully concentrated to a maximum of 1.5 mg/ml using Amicon Ultra-15 50 kD units.
- PBS-equilibrated PD-10 desalting columns were used to change the buffer to PBS, followed by another protein concentration measurement at 280 nm.
- the 96-well Microlon microtiter plates were purchased from Greiner Bio-One (Frickenhausen, Germany) .
- Recombinant human IL- 6 and soluble IL-6 receptor (sIL-6R) were obtained from BioSource (Solingen, Germany) and R&D Systems (Wiesbaden, Germany), respectively.
- the primary anti-sIL-6R antibody (clone M91, mouse IgGl) was from Beckman Coulter (Krefeld, Germany) .
- the secondary anti-mouse IgG HRP-conjugated antibody from GE Healthcare (Freiburg, Germany) was the same as in the Western blotting experiments (see Example 1, A and C) .
- Ready- to-use Tetramethylbenzidin (TMB) HRP substrate was purchased from Sigma-Aldrich (Taufkirchen, Germany) .
- sgpl30Fc variants For quality control and comparison between different lots of sgpl30Fc variants, a standard ELISA was designed. All steps except for coating were carried out at room temperature, all washing steps were performed three times with a volume of 250 ⁇ l, and all conditions were measured in triplicate. A 96 well microtiter plate was coated with 100 ng/Well of original sgpl30Fc as an internal standard and an equimolar quantity of sgpl30 (D1-D3) S n -Fc in 100 ⁇ l PBS at 4 0 C overnight. Thus, sgpl30Fc and its variants served as capture reagent, while the bound sIL- ⁇ R was detected (see below) .
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AT06841152T ATE471945T1 (de) | 2005-12-23 | 2006-12-22 | Als medikament geeignete lösliche varianten des gp130-moleküls |
US12/158,285 US7851182B2 (en) | 2005-12-23 | 2006-12-22 | Soluble GP130 molecule variants useful as a medicament |
CA002633213A CA2633213A1 (en) | 2005-12-23 | 2006-12-22 | Soluble gp130 molecule variants useful as a medicament |
AU2006328909A AU2006328909A1 (en) | 2005-12-23 | 2006-12-22 | Soluble gp130 molecule variants useful as a medicament |
DE602006015103T DE602006015103D1 (de) | 2005-12-23 | 2006-12-22 | Als medikament geeignete lösliche varianten des gp130-moleküls |
JP2008546287A JP5390191B2 (ja) | 2005-12-23 | 2006-12-22 | 医薬として有用な可溶性gp130分子変異体 |
EP06841152A EP1994053B1 (de) | 2005-12-23 | 2006-12-22 | Als medikament geeignete lösliche varianten des gp130-moleküls |
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EP05028420A EP1801121A1 (de) | 2005-12-23 | 2005-12-23 | Lösliche Varianten des Moleküls gp130 nützlich als Arzneimittel |
EP05028420.7 | 2005-12-23 |
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EP (2) | EP1801121A1 (de) |
JP (1) | JP5390191B2 (de) |
AT (1) | ATE471945T1 (de) |
AU (1) | AU2006328909A1 (de) |
CA (1) | CA2633213A1 (de) |
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Cited By (3)
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JP2011501944A (ja) * | 2007-10-19 | 2011-01-20 | コナリス リサーチ インスティチュート アーゲー | 改善された結合活性をもつ可溶性gp130変異蛋白質 |
US10519218B2 (en) | 2014-12-01 | 2019-12-31 | Ferring B.V. | Selective IL-6-trans-signalling inhibitor compositions |
US11198721B2 (en) | 2014-12-01 | 2021-12-14 | Ferring B.V. | Methods for treatment of inflammatory disease or IL-6-mediated condition with gp130 protein |
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DE602006016765D1 (de) | 2006-06-30 | 2010-10-21 | Conaris Res Inst Ag | Verbesserte sgp 130Fc Dimere |
JP5424330B2 (ja) * | 2007-07-26 | 2014-02-26 | 国立大学法人大阪大学 | インターロイキン6受容体阻害剤を有効成分とする眼炎症疾患治療剤 |
WO2017106244A1 (en) * | 2015-12-14 | 2017-06-22 | The Trustees Of The University Of Pennsylvania | Compositions and methods for regulatable antibody expression |
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WO1994012520A1 (en) * | 1992-11-20 | 1994-06-09 | Enzon, Inc. | Linker for linked fusion polypeptides |
EP1148065A1 (de) * | 2000-04-21 | 2001-10-24 | Stefan Rose-John | Fusionsproteine, die zwei lösliche gp130 Moleküle enthalten |
EP1491554A1 (de) * | 2003-06-23 | 2004-12-29 | CONARIS research institute AG | PEGlierte lösliche gp130-Dimeren geignet als Medikament |
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AU778998B2 (en) * | 1998-09-22 | 2004-12-23 | University Of Maryland At Baltimore | Cystine knot growth factor mutants |
CN1341121A (zh) * | 1999-01-07 | 2002-03-20 | 利思进药品公司 | 作为Fc融和蛋白之抗肥胖蛋白质的表达和外运 |
ATE384783T1 (de) * | 2001-10-19 | 2008-02-15 | Zymogenetics Inc | Dimerisierter wachstumsfaktor sowie materialien und verfahren zu seiner herstellung |
JP2008504012A (ja) * | 2004-01-28 | 2008-02-14 | シントニックス・ファーマシューティカルズ・インコーポレーテッド | 不妊治療のためのへテロ二量体卵胞刺激ホルモン−Fc(FSH−Fc)融合タンパク質 |
-
2005
- 2005-12-23 EP EP05028420A patent/EP1801121A1/de not_active Withdrawn
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- 2006-12-22 AU AU2006328909A patent/AU2006328909A1/en not_active Abandoned
- 2006-12-22 DE DE602006015103T patent/DE602006015103D1/de active Active
- 2006-12-22 US US12/158,285 patent/US7851182B2/en active Active
- 2006-12-22 AT AT06841152T patent/ATE471945T1/de not_active IP Right Cessation
- 2006-12-22 WO PCT/EP2006/012515 patent/WO2007071449A1/en active Application Filing
- 2006-12-22 ES ES06841152T patent/ES2347694T3/es active Active
- 2006-12-22 JP JP2008546287A patent/JP5390191B2/ja active Active
- 2006-12-22 CA CA002633213A patent/CA2633213A1/en not_active Abandoned
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WO1994012520A1 (en) * | 1992-11-20 | 1994-06-09 | Enzon, Inc. | Linker for linked fusion polypeptides |
EP1148065A1 (de) * | 2000-04-21 | 2001-10-24 | Stefan Rose-John | Fusionsproteine, die zwei lösliche gp130 Moleküle enthalten |
EP1491554A1 (de) * | 2003-06-23 | 2004-12-29 | CONARIS research institute AG | PEGlierte lösliche gp130-Dimeren geignet als Medikament |
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2011501944A (ja) * | 2007-10-19 | 2011-01-20 | コナリス リサーチ インスティチュート アーゲー | 改善された結合活性をもつ可溶性gp130変異蛋白質 |
US10519218B2 (en) | 2014-12-01 | 2019-12-31 | Ferring B.V. | Selective IL-6-trans-signalling inhibitor compositions |
US11198721B2 (en) | 2014-12-01 | 2021-12-14 | Ferring B.V. | Methods for treatment of inflammatory disease or IL-6-mediated condition with gp130 protein |
US11306136B2 (en) | 2014-12-01 | 2022-04-19 | Ferring B.V. | Selective IL-6-trans-signalling inhibitor compositions |
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DE602006015103D1 (de) | 2010-08-05 |
EP1994053B1 (de) | 2010-06-23 |
EP1994053A1 (de) | 2008-11-26 |
EP1801121A1 (de) | 2007-06-27 |
CA2633213A1 (en) | 2007-06-28 |
JP2009520480A (ja) | 2009-05-28 |
JP5390191B2 (ja) | 2014-01-15 |
AU2006328909A1 (en) | 2007-06-28 |
US20090227499A1 (en) | 2009-09-10 |
US7851182B2 (en) | 2010-12-14 |
ATE471945T1 (de) | 2010-07-15 |
ES2347694T3 (es) | 2010-11-03 |
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